White matter damage is an essential feature of stroke, traumatic brain injury and perinatal hypoxia/ischemia. Oligodendrocytes (OLs) are the myelin-forming glial cells of the brain, thus OL damage has profound consequences for the function of white matter. Our long-term goal is to reveal, in-depth, the mechanisms of OL damage for the design of novel and more effective therapeutic strategies. The focus of this project is on a novel mechanism of mitochondrial dysfunction leading to apoptotic OL death after brain ischemia/reperfusion (IR). A hallmark of OL metabolism is the generation of large quantities of sphingolipids which are key lipid components of myelin. In addition to their structural function, bioactive sphingolipids play fundamental roles in the regulation of proliferation and apoptosis. Ceramide, a pro-apoptotic sphingolipid, is tightly regulated in cells and its participation in cell death signaling pathways is controlled by a rapid conversion of ceramide into less deleterious sphingolipids, including sphingosine-1-phosphate, a pro-survival sphingolipid. Sirtuins, a family of protein deacetylases, are important regulators of metabolism and longevity. Sirtuin 3 (SIRT3) coordinates the adaptive responses of several metabolic pathways in mitochondria. Our preliminary data indicate that SIRT3 is involved in the reciprocal regulation of bioactive sphingolipid metabolites that ultimately results in OL apoptosis. Our goals are to define how SIRT3 participates in the apoptotic program of OLs after cerebral IR and to unravel how SIRT3 could be manipulated for preventive and therapeutic purposes. Our central hypothesis is that cerebral IR triggers SIRT3- mediated, ceramide-dependent OL apoptosis and that sphingosine-1-phosphate is a key regulator of SIRT3 activity in OLs. Our Specific Aims are: (1) Identify the mechanisms of SIRT3-mediated OL apoptosis; (2) Determine the mechanisms regulating SIRT3 activity in OLs. We will use a multi-disciplinary and integrative approach, combining in vitro and in vivo studies in transgenic and knockout mouse stroke models with modern pharmacological, biochemical and bioenergetics methodologies. To monitor mitochondrial function in live OLs, a laser scanning confocal/multi-photon microscope equipped with femtosecond laser and META spectral detection will be used. Accurate quantification of multiple natural sphingolipid species will be achieved by a state-of-the-art mass spectrometry-based methodology. At the conclusion of this research program, we should establish a mechanistic link between SIRT3, the key regulator of mitochondrial metabolic pathways, and mitochondrial dysfunction leading to OL apoptosis. These studies will reveal novel determinants of cell demise in stroke which are highly likely to serve as new targets for the rational design of more effective therapies. The proposed studies will generate a coherent and unified view of SIRT3's role in the mitochondrial pathobiology of OLs and this knowledge will benefit the research in stroke, traumatic brain injury, perinatal hypoxia/ischemia and chronic neurological disorders characterized by OL loss.